Heart failure kills 1 in 10 people over the age of 65 in the USA. We recently identified a combination of four cell- cycle regulators that induces stable cytokinesis in adult cardiomyocytes to replace their loss following myocardial infarction. Specifically, adenoviral overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 (collectively known as 4F, i.e. four factors), efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome- mediated degradation of the protein products in cardiomyocytes. In vivo, lineage tracing using the Mosaic Analysis of Double Marker (MADM) mouse model revealed that 15?20% of adult cardiomyocytes expressing the 4F underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Currently, our approach is the most robust method to induce cardiomyocyte proliferation; however, clinical applicability in humans is limited by concerns for tumorigenic potential in other organs. Our preliminary data in vitro shows that in other cell types (e.g. neurons), 4F induce cell proliferation continuously for 5-6 successive rounds over 7 days. Therefore, in this proposal we will focus on approaches to make cell cycle induction in cardiomyocytes more clinically applicable and avoid any oncogenic potential. We hypothesize that a transient and cardiomyocyte-specific expression is needed to induce one cycle of cardiomyocyte proliferation to avoid any potential adverse effects on other tissues. Our preliminary data shows that TNNT2 derived 4F expression in NIL is robustly inducing proliferation in vitro and in vivo and improved cardiac function after myocardial infarction. Here, we will rigorously demonstrate the efficacy and safety of this polycistronic NIL encoding 4F expression driven by the cardiac specific promoter of TNNT2 (TNNT-4F-NIL) in inducing cardiomyocyte division and improving cardiac function after myocardial infarction in vivo in rat and pig models as well as in situ in human heart slices from healthy and infarcted hearts. Here, we aim first to fully demonstrate the efficacy and specificity of TNNT-4F-NIL to induce proliferation only in cardiomyocytes in vivo. Secondly, we will test the functional efficacy and perform initial safety studies for TNNT-4F-NIL in rat and pig models of heart failure. Lastly, we will demonstrate the efficacy of TNNT-4F-NIL in inducing adult human cardiomyocyte proliferation in situ in human heart slices and improving contractile function of failing human heart slices. This study will address all preclinical efficacy testing and perform initial safety studies for one of the most promising approaches to regenerate the heart. The successful completion of this study will allow the start of first in human clinical trial.